Circadian (daily) rhythms are a crucial component of human health that regulates sleep, alertness, hormones, metabolic activities of various tissues, and many other biological processes. The ultimate explanation for the mechanism of circadian oscillators will require characterizing the structures, functions, and interactions of the molecular components of these clocks. The current project is to elucidate the basic principles of circadian clocks at a biophysical/molecular level in a model system, the prokaryotic cyanobacteria, where genetic/biochemical studies have identified three key clock proteins, KaiA, KaiB, and KaiC. These three proteins can reconstitute a circadian oscillator in vitro. This remarkable observation has led to a re-evaluation of our understanding of circadian clocks in all organisms, including mammals. The crystal structure of the KaiA, KaiB, and KaiC proteins have been reported-these are the first clock proteins to have their 3-D structures determined. The advent of atomic resolution structures of the molecular components of a clockwork marks a dramatic watershed in circadian research by ushering in truly molecular analyses of circadian mechanisms. The current project will determine the molecular basis of the core clockwork by biophysical, genetic, and structural approaches. In particular, the relative roles of (i) the rhythmic formation of a KaiA/KaiB/KaiC complex as compared with (ii) the rhythm of KaiC phosphorylation as the key cogs in the timing mechanism will be assessed. The biophysical/molecular analyses will be coupled with mathematical modeling to determine the essential parameters. Temperature compensation of this biological clock will be investigated by screening for temperature dependent mutants in vivo followed by in vitro analyses. Finally, the linkage between this core clockwork and the global orchestration of gene expression over the daily cycle will be illuminated by testing the novel "oscilloid" model, which proposes that the core clockwork rhythmically regulates promoter activity by modulating chromosomal topology as a function of circadian time. RELEVANCE TO PUBLIC HEALTH: This project will clarify circadian mechanisms at molecular levels that were heretofore unattainable. Biological clocks have been found to be crucial for mental health. In addition, biological clocks are key for optimal performance under shiftwork and "jet-lag" conditions that affect a large proportion of the USA workforce. Knowledge of the circadian mechanism along with the development of therapies to properly phase sleep will allow us to enhance the performance, alertness, and well-being of shiftworkers and travelers in addition to improving the quality of life for depressed subjects.